The Mast Cell-restricted Tryptase mMCP-6 Has a Critical Immunoprotective Role in Bacterial Infections

Abstract

Although it has been shown that mast cell-deficient mice have diminished innate immune responses against bacteria, the most important immunoprotective factors secreted from activated mast cells have not been identified. Mouse mast cell protease 6 is a tetramer-forming tryptase. This serine protease is abundant in the secretory granules and is exocytosed upon bacterial challenge. Here we have described the generation of a mast cell protease-6-null mouse. Our discovery that mice lacking this neutral protease cannot efficiently clear Klebsiella pneumoniae from their peritoneal cavities reveals an essential role for this serine protease, and presumably its human ortholog, in innate immunity.Approximately 50% of the weight of a mature tissue mast cell (MC) 2 consists of protease-serglycin proteoglycan complexes stored in the secretory granules. In humans, ␤ tryptases are the most abundant MC-restricted neutral proteases (1-3). The corresponding tryptases in mice are mouse MC protease (mMCP)-6 (4, 5) and mMCP-7 (6), with mMCP-6 being the most similar in amino acid sequence and substrate specificity to human tryptase (hTryptase) ␤1 (7-9). MCs are the only cells that express mMCP-6, and this serine protease is particularly abundant in those MCs that reside in the peritoneal cavity, skin, and lung (4,5,10).Numerous biochemical studies have been carried out to understand the biosynthesis and substrate preference of mMCP-6. This tryptase is initially translated as a zymogen with a 245-mer mature domain. When the signal and propeptides are proteolytically removed, the mature protease spontaneously forms tetramers with the active site of each monomer facing the central core of the tetramer unit, as first described for its human ortholog (11). A positively charged face forms on the surface of each monomer, thereby allowing mature mMCP-6 to interact with negatively charged serglycin proteoglycans in the Golgi complex. The resulting tryptase-serglycin macromolecular complexes are then targeted and packaged in the cell secretory granules. When exocytosed, these complexes are retained in connective tissues for hours because of their large sizes (12). Protease inhibitors are abundant in blood. Nevertheless, no circulating protease inhibitor has been identified that rapidly inactivates mMCP-6 or hTryptase ␤1. Substrate specificity studies carried out using varied peptide combinatorial libraries revealed that recombinant mMCP-6 (7) and hTryptase ␤1 (8, 9) prefer to cleave peptides having a Pro at residues P2 to P5 and a Lys or Arg at residue P1. However, due to the unique structural constraints of the tetramer unit, the abilities of mMCP-6 and hTryptase ␤1 to cleave large-sized proteins are very limited. Thus, the importance of these evolutionally conserved enzymes in MC-dependent reactions remains to be determined.MC development in vivo is highly dependent on the cytokine kit ligand/stem cell factor on the surface of mesenchymal cells and its tyrosine kinase receptor c-Kit/CD117 on the surface of MC-committed progenitors. Signaling...

The mouse mast cell–restricted tetramer‐forming tryptases mouse mast cell protease 6 and mouse mast cell protease 7 are critical mediators in inflammatory arthritis

“…In more recent studies, we created a C57BL/6 mouse strain that expresses mMCP-7 but not mMCP-6 (34). We also created another C57BL/6 mouse strain that lacks both tryptases (35). Using these NDST-2 Ϫ , mMCP-6 ϩ /mMCP-7 Ϫ , mMCP-6 Ϫ /mMCP-7 ϩ , and mMCP-6 Ϫ /mMCP-7 Ϫ C57BL/6 mice, we now show that MC-restricted tryptase-heparin complexes play prominent roles in the model of mBSA/IL-1␤-induced arthritis.…”

“…Finally, our data raise the possibility that inhibition of the ␤ tryptases in MCs could have therapeutic value in the treatment of RA. However, on a cautionary note, the finding that our mMCP-6-deficient mice cannot efficiently combat bacterial (35) and helminthic (34) infections raises the possibility that patients with RA might be more susceptible to infectious organisms if their MC-restricted tryptases are inactivated systemically.…”

“…Mouse MCP-6 and its human homolog hTryptase-␤1 induce neutrophil recruitment when injected into the mouse lung or peritoneal cavity (41,46). Moreover, transgenic mice lacking mMCP-6 have an impaired defense against bacterial infections due to a markedly reduced ability to recruit neutrophils into the infected tissue site (35). The discovery that recombinant mMCP-6 and hTryptase-␤1 can induce increased expression of CXCL8-like chemokines by cultured cells (41,47) suggests that these serine proteases promote the accumulation of neutrophils ( Figures 1C and 3C) in the rheumatoid joint by inducing bystander cells to increase markedly their production of chemokines that are recognized by the granulocytes.…”

“…Using a homologous recombination knockout/knockin approach, we recently created a transgenic C57BL/6 mouse strain that lacks mMCP-6 but expresses mMCP-7 (34). We also recently created a transgenic C57BL/6 mouse strain that lacks both tryptases, using another homologous recombination approach (35). These NDST-2 Ϫ , mMCP-6 ϩ / mMCP-7 Ϫ , mMCP-6 Ϫ /mMCP-7 ϩ , and mMCP-6 Ϫ / mMCP-7 Ϫ C57BL/6 mice now allow us the opportunity to evaluate the roles of MC-restricted tryptase-heparin complexes in a manner that previously was not possible.…”

Immunomodulatory mast cells: negative, as well as positive, regulators of immunity

“…If that mediator is selectively expressed by mast cells, and if its deletion does not significantly influence the expression of other mastcell products, then it is possible to draw conclusions about the role of that mast-cell product in vivo. For example, mice that lack mouse mast-cell protease-1 (mMCP-1) [26][27][28] , mMCP-4 29,30 , mMCP-6 [31][32][33] , or mouse mast cell-carboxypeptidase A (mMC-CPA) 34 , or that have a mutated mMC-CPA that essentially lacks enzymatic activity 11 , have been used to analyze whether the absence of these proteases (or their enzymatic activity) influences other aspects of the mast-cell phenotype, such as content of other stored mediators, as well as to define the functions of such mast-cell-associated proteases in vivo.…”